Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to
Guideline:
other: EPA, Health Effects Test Guidelines; OPPTS 870.3550: Reproduction/Developmental Toxicity Screening Test, (Jul 2000)
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: solid, violet
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: (P) 11-12 wks
- Weight at study initiation: (P) Males: about 330 g; Females: about 200 g
- Housing: individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²), with the following exceptions: during overnight matings, male and female mating partners were housed together in Makrolon type M III cages; pregnant animals and their litters were housed together until PND 4 (end of lactation).
- Diet: Kliba maintenance diet mouse-rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): of 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water was filled up to the desired volume, subsequently released with a high speed homogenizer. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer. The test substance preparations were produced at least once a week.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyses of the test substance preparations were carried out as a separate study at the test facility Competence Center Analytics of BASF SE, 67056 Ludwigshafen, Germany, under the responsibility of a Study Director of this test facility. The study was carried out in compliance with the Principles of Good Laboratory Practice.
The stability of the test substance in drinking water was demonstrated over a period of 7 days at room temperature. As the mixtures were stored no longer than this time period, the stability was guaranteed. The samples were homogenous within the different levels of concentration. The concentration control analyses of all concentrations revealed that the values were in the expected range of the target concentrations, i.e. were always in a range of about 90-110% of the nominal concentrations. These results demonstrate the correctness of the concentrations of the test article.
Details on mating procedure:
In general, each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same test group.
The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 07.00 - 09.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm was detected was denoted gestation day (GD) 0 and the following day "GD 1".
Duration of treatment / exposure:
The duration of treatment covered premating period of 2 weeks and a mating period (max. of 2 weeks) in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females.
Frequency of treatment:
daily
Duration of test:
until PND 4
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
MORTALITY
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.

CLINICAL OBSERVATIONS: Yes
A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each affected animal.
The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis.
On weekdays (except public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.
The day of littering was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.

BODY WEIGHT: Yes
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice.
The body weight change of the animals was calculated from these results.
The following exceptions are notable for the female animals:
• During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition (PND 0) and on PND 4.
• Females without litter, waiting for necropsy, were weighed weekly. These body weight data were solely used for the calculations of the dose volume; therefore these values are only documented in the Individual Tables (PART II).
• Females between PND 4 and sacrifice were weighed once a week.

FOOD CONSUMPTION:
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
• Food consumption was not determined during the mating period (male and female F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined on GD 0, 7, 14 and 20.
• Food consumption of F0 females, which gave birth to a litter was determined on PND 1 and PND 4.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel) and in males after the premating period.

PATHOLOGY
Necropsy:
All animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.

Organ weights:
Weight assessment was carried out on all animals. The following weights were determined:
1. Anesthetized animals
2. Epididymides
3. Testes

Histopathology:
The following organs / tissues of all parental animals were preserved in neutral-buffered 4% formaldehyde or in modified Davidson’s solution:
All gross lesions, Cervix, Coagulating gland, Epididymides (modified Davidson’s solution), Ovaries (modified Davidson’s solution), Oviducts, Prostate gland, Seminal vesicles, Testes (modified Davidson’s solution), Vagina, Uterus.
After the organs were fixed, processing, examination by light microscopy and evaluation of findings were performed. The following tissues were examined: testes, epididymides, ovaries from all control and high dose animals.
Fetal examinations:
External examinations: Yes

Viability/mortality:
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays.
The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1 - 4 (lactation period) were determined. Pups which died accidentally or were sacrificed due to maternal death were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation day 4

Sex ratio:
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. The sex of the pups was finally confirmed at necropsy.

Clinical observations:
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.

Pup body weight data:
The pups were weighed on the day after birth (PND 1) and on PND 4. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning). “Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.

Pathology:
All pups with scheduled sacrifice on PND 4 were sacrificed under isoflurane anesthesia with CO2. All pups were examined externally and eviscerated; their organs were assessed macroscopically.
All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically.
All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.
Statistics:
The following statistical methods were used:
- Comparison of the dose group with the control group was performed using the Student’s t-test (two-sided) for the hypothesis of equal means (Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), gestation days, implantation sites, pups delivered, life pups day x)
- Comparison of the dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions (Male and female mating indices, male and female fertility indices, gestation index, females delivering, females with liveborn pups, females with stillborn pups, females with all stillborn pups)
- Comparison of the dose group with the control group was performed using the WILCOXON test (one-sided+) for the hypothesis of equal medians (Mating days until day 0 pc)
- Comparison of the dose group with the control group was performed using the WILCOXON test (one-sided-) for the hypothesis of equal medians (Viability Index)
- Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pair wise comparison of each dose group with the control group was performed using the WILCOXON test for the hypothesis of equal medians (Weight parameters)
Indices:
male and female mating index, male and female fertility index, gestation index, postimplantation loss, Live birth index, pup number and status at delivery, viability index

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
see 7.8.1
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
see 7.8.1
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
see 7.8.1

Maternal developmental toxicity

Number of abortions:
not examined
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The postimplantation loss was 8.86% in test group 0, 7.90% in test group 1, 1.78% in test group 2 and 4.28% in test group 3.
These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
Total litter losses by resorption:
not examined
Early or late resorptions:
not examined
Dead fetuses:
no effects observed
Description (incidence and severity):
The liveborn birth index was 98.8% in the control group, 96.5 in test group 1, 99.2 in test group 2 and 99.1% in test group 3. These values reflect the normal range of biological variation inherent in the strain of rats used for this study.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
The mean number of delivered F1 pups was 10.6 in test group 0, 12.7 in test group 1, 13.1 in test group 2 and 12.7 in test group 3.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw (total dose)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw (total dose)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, non-treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the control group.
Two male runts were seen in test group 0. Each one female runt was seen in test group 1 and 2 (100 and 300 mg/kg bw/d).
These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The liveborn birth index was 98.8% in the control group, 96.5 in test group 1, 99.2 in test group 2 and 99.1% in test group 3. These values reflect the normal range of biological variation inherent in the strain of rats used for this study.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
The viability index indicating pup mortality during lactation (PND 0 - 4) was 100% in all test groups.
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
One male and one female F1 pup of test group 1 showed post mortem autolysis. Each one female F1 pup of test group 2 and 3 (300 and 1000 mg/kg bw/d) showed an empty
stomach. This finding was assessed as being spontaneous in nature and without biological relevance.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no teratogenic effects, no developmental toxicity

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of this reproduction/developmental toxicity screening test the NOAEL (no observed adverse effect level) for developmental toxicity was concluded to be 1000 mg/kg bw/day for maternal animals and offspring. The test substance did not show abnormalities in offspring and thus, was not considered to be teratogenic.
Executive summary:

In a GLP-compliant Reproduction/Developmental Toxicity Screening Test according to OECD guideline 421, the test article was administered orally via gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 mg/kg bw/d (test group 0), 100 mg/kg bw/d (test group 1), 300 mg/kg bw/d (test group 2) and 1000 mg/kg bw/d (test group 3). The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances. F0 animals were mated 13 days after the beginning of treatment to produce a litter (F1 generation pups). F0 animals were examined for their reproductive performance including determinations of the number of implantations and the calculation of the postimplantation loss in all F0 females. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1 and PND 4 and their viability was recorded. At necropsy on PND 4, all pups were sacrificed under isoflurane anesthesia with CO2and examined macroscopically for external and visceral findings at necropsy. All F0 parental animals were sacrificed by decapitation under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed. No signs of general systemic toxicity were observed in male or female parental animals of all test groups (100, 300 and 1000 mg/kg bw/d) during the entire study period. All male and female animals of test group 2 (300 mg/kg bw/d) and 3 (1000 mg/kg bw/d) showed black discolored feces from study day 1 until the end of the study. Regarding pathology, macroscopically black discoloration of the content of the digestive tract in numerous animals was observed. Beside the discoloration no signs of toxicity in the respective tissues were noted macroscopically. This finding is regarded to be a consequence to the oral intake of the violet test substance and therefore treatment related but not adverse in nature. Regarding fertility and reproductive performance, no signs of toxicity were observed in male or female parental animals of all test groups during the entire study. Thus, under the conditions of this modified reproduction/developmental toxicity screening test the NOAEL (no observed adverse effect level) for developmental toxicity in the F1 progeny was found to be 1000 mg/kg bw/day. The NOAEL for general, systemic toxicity was 1000 mg/kg bw/day.